The long term goals of this project are to determine the functional roles of small, non-coding microRNAs (miRNAs) in regulating cartilage development and homeostasis. Acquiring such new knowledge will be critical for the generation of alternative microRNA-based strategies to treat trauma-induced cartilage defects and prevent progression toward osteoarthritis, the most common degenerative disease in humans. To elucidate which miRNAs to study further, we carried out TaqMan(R)-based microarrays on RNA extracted from human embryonic cartilage tissue to identify highly-expressed miRNAs as well as differentially- expressed miRNAs between precursor, differentiated and hypertrophic chondrocytes in vivo. From these studies, we have generated an exciting candidate list of miRNAs to pursue that has not yet been studied within the context of cartilage biology. We hypothesize that these miRNAs are functional in regulating specific phases of chondrocyte differentiation and/or are involved in modulating homeostasis of mature cartilage tissue under pathophysiological conditions. In Specific Aim 1, we will determine the function of miRNAs in regulating phases of chondrocyte differentiation by utilizing a human mesenchymal stem cell (MSC) assay system in vitro and murine embryonic metatarsal bone explants ex vivo. A state-of-the-art approach to immunoprecipitate RNA- induced silencing complexes (RISCs) followed by RNA sequencing will be applied to identify miRNA target genes and the cellular pathways affected. In Specific Aim 2, a well-controlled, non-invasive murine joint loading model will be used to determine how expression levels of specific miRNAs in chondrocytes are altered in response to pathophysiological conditions. The function of these miRNAs in regulating catabolic or anabolic pathways in chondrocytes will also be elucidated. Clinically, the research proposed in this application is important given that there is an unmet need for: 1) effective strategies to repair or regenerate articular cartilage using stem cells and 2) therapeutic interventions to treat injured articular cartilage, control homeostasis and prevent development of post-traumatic osteoarthritis. Since miRNAs can fine-tune the expression of multiple genes, they may be more useful tools than siRNA in complex diseases such as osteoarthritis that involve interactomes of molecular players. Notably, the studies proposed in this application are timely given the emergence of miRNAs as potential therapeutic targets in other disease scenarios in addition to current advances being made to deliver small molecules to mimic or inhibit miRNA function in vivo.